Pancreatic cancer is one of the deadliest cancers, with a mortality rate of nearly 100%. The vast majority of pancreatic cancers are discovered too late to resect and do not respond to current chemotherapeutic regimens. Because surgery generally has little to offer curatively, medical management of pancreatic cancer has been a growing area of exploration. The long-term objective of this R21 pilot project application is to determine whether the use of agents that enhance cellular ceramide levels will be a viable approach for treating pancreatic cancer. Some drugs, especially in combination with certain other drugs, selectively kill cancers by stimulating over-production of normal cellular waxes known as ceramides. This area of investigation in pancreatic cancer is unexplored. Ceramides activate intrinsic cell death cascades that promote downstream generation of reactive oxygen species (ROS), caspase activity, expression of proapoptotic Bim (Bcl-2-interacting mediator of death), and downregulate survivin. These events contribute to apoptosis, autophagy, and cellular senescence. To determine whether targeting ceramide metabolism is an effective avenue for treating pancreatic cancer, two agents that promote ceramide formation in pancreatic cancer cells will be studied. These agents are fenretinide (4-HPR), a vitamin A analog, and Valspodar (PCS 833), a cyclosporin A analog. This study will employ cultured human pancreatic cancer cell lines and an SV-40- transformed, immortalized pancreatic cell line. The overall hypothesis is that pancreatic cancer cells are acutely sensitive to ceramide with the corollary to this hypothesis being that enhancing either the level of ceramide or specific molecular species of ceramide propels cytotoxicity along specific routes. This study has three specific aims designed to assess pancreatic cancer cell vulnerability to ceramide and ceramide-governed cell death cascades. Specific Aim 1 will characterize pancreatic cancer cell response to ceramide by using C6- ceramide, a short-chain, cell permeable analog of natural ceramide, and determine if cytotoxic responses are enhanced by the introduction of enzyme inhibitors that block ceramide metabolism. Specific Aim 2 will characterize the effect of 4-HPR and PSC 833 on cell viability and determine if cytotoxicity is linked to ceramide. Included here are mass spectroscopic analysis of dihydroceramides and ceramides generated in response to 4-HPR and PSC 833 to determine whether specific molecular species provoke specific responses, autophagy and senescence for example. Specific Aim 3 will assess the influence of ceramide generated in response to 4-HPR and PSC 833 on biochemical events associated with activation of intrinsic cell death. The innovative nature of this approach is that ceramide pathways can be manipulated, presenting a ready platform to improve efficacy. The very limited efficacy of available therapies for pancreatic cancer and the very high incidence of treatment failure are strong reasons to pursue new approaches. PUBLIC HEALTH RELEVANCE: This project will test a new approach to treat pancreatic cancer by studying the influence of a non-toxic vitamin A analog (4-HPR). 4-HPR can selectively kill cancer cells by stimulating over-production of normal cell waxes, called ceramides, which in excess are lethal to malignant cells and not normal cells. Because pancreatic cancer cells can metabolize ceramide to non-toxic byproducts, we will investigate inclusion of "partnering drugs" that block ceramide metabolism in order to fine-tune this novel approach to therapy.